Immunoglobulins are high molecular weight proteins which fall into five major classes: IgA, IgD, IgE, IgG and IgM. Collectively, these proteins are commonly referred to as antibodies. Antibodies form, for nearly all higher organisms, the basis of a fundamental immunological defense system against a variety of pathological insults.
A characteristic property of antibodies, regardless of class, is that they function in their defense roles by forming specific complexes with portions of the invading pathogen. This feature of antibodies has been exploited in-vitro for a large variety of analytical testing applications such as Radio Immuno Assay (RIA), and Enzyme Linked Immunoassay (ELISA). In-vivo, the specific binding properties of antibodies has been exploited in a large variety of immuno-therapy and imaging techniques.
The medical and commercial importance of antibodies has resulted in a variety of techniques designed to isolate and purify antibodies of a particular class.
A particularly useful method for IgG isolation involves the use of immobilized Protein-A in an affinity chromatographic format. (U.S. Pat. No. 3,995,018 to Sjoquist). Protein A is a bacterial wall derived protein from Staphylococcus aureus or derived more conveniently via recombinant techniques. A deficiency inherent in the use of protein-A for IgG isolation is that preparations are frequently contaminated with antibodies from the IgM and IgA class. Protein G, a bacterial wall protein derived from group G Streptococci, has also enjoyed utility as an IgG binding protein and has been shown to show greater specificity toward IgG than does protein A.
Jacalin, an alpha-D-galactose binding lectin extracted from jack-fruit seeds (Artocarpus integrifolia) has been reported to possess bio-specific binding properties for human IgA (Kumar, et al., J. Biosci. 4, p 257-261 (1982)). By use of immobilized jacalin, human IgA can be prepared which is free of contaminating IgG or IgM.
Unfortunately, very little has been available to the investigator with which to pursue practical methods for the isolation of IgM. This paucity of methodology is in spite of the fact that IgM is an extremely valuable class of immunoglobulin. For example, with many antigenic challenges, IgM is the only class of antibody produced by the host animal.
Recently, Nethery, et al., in the Journal of Immunological Methods, 126 (1990) p 57-60 described a single-step affinity chromatographic method for purification of IgM based upon a temperature dependant interaction of immobilized protein C1q with IgM. The method for IgM purification based on C1q represents a marked improvement over what has been heretofore described. However, as recognized by Nethery, et al., C1q binds not only to IgM, but also IgG. C1q has been reported to have substantial binding affinity for monomeric IgG based upon observations by Sledge and Bing, J. Biol. Chem. 248, 2818-2823 (1973). The result of binding ability of C1q for IgG is that preparations of IgM based upon a C1q affinity matrix may be adulterated with various and sporadic levels of IgG. Thus, an object of the instant invention is to provide an efficient isolation process which can be used to obtain IgM in substantially pure form uncontaminated by IgG.